CircSLC22A3 inhibits the invasion and metastasis of ESCC via the miR-19b-3p/TRAK2 axis and by reducing the stability of m6A-modified ACSBG1 mRNA

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Abstract
Background: Esophageal squamous cell carcinoma (ESCC) is a major contributor to cancer-related deaths, driven by its invasive and metastatic nature. Circular RNAs (circRNAs) are increasingly recognized as regulators of cancer progression, primarily through miRNA sponging and interactions with RNA-binding proteins. Their dysregulation has been linked to the development of in various cancers. The present study aimed to investigate the potential involvement of circSLC22A3 in the pathogenesis of ESCC. Methods: CircSLC22A3 expression in ESCC tissues and cells was analyzed using transcriptome sequencing and RT-qPCR. Its circular structure was validated through Sanger sequencing, agarose gel electrophoresis, RNase R digestion, and random priming assays. Subcellular localization was determined by nucleoplasmic separation and fluorescence in situ hybridization (FISH). Clinical correlations were assessed via tissue microarrays. Functional roles of circSLC22A3 in ESCC progression were investigated through in vitro and in vivo assays. Downstream miR-19b-3p and target gene TRAK2 were screened by bioinformatics analysis and RT-qPCR, with binding confirmed via luciferase reporter assays. RNA pulldown combined with RNA immunoprecipitation (RIP) identified IGF2BP1 as a circSLC22A3-interacting protein. RNA-seq and RT-qPCR revealed ACSBG1 as a key downstream effector. IGF2BP1-mediated m6A modification of ACSBG1 was mapped by MeRIP-seq and RIP, with mRNA stability assessed via Actinomycin D assay. ACSBG1 expression and biological function in ESCC were confirmed by immunohistochemistry, RT-qPCR, and functional assays. Results: Significant downregulation of circSLC22A3 was observed in both ESCC tissues and cell lines. Overexpression of circSLC22A3 significantly reduced ESCC cells’ migration and invasion capabilities. Mechanistic investigation revealed that circSLC22A3 played a pivotal role in the invasion and metastasis of esophageal cancer through distinct pathways. On one hand, circSLC22A3 functioned as a miR-19b-3p sponge to augment trafficking kinesin protein 2 (TRAK2) expression, while, on the other hand, circSLC22A3 formed a protein-RNA complex with IGF2BP1, resulting in the degradation of acyl-CoA synthetase bubblegum family member 1 (ACSBG1) mRNA through the recognition of m6A modification, thereby suppressing invasion and metastasis of ESCC. Conclusions: The present study identified circSLC22A3 as a new tumor suppressor that inhibited ESCC progression through both the circSLC22A3/ miR-19b-3p/ TRAK2 and circSLC22A3/ IGF2BP1/ ACSBG1 axes.
Funding Information
  • The Fund of North Sichuan Medical College (CBY23-ZDA11, CBY23-ZDA11)
  • Sichuan Provincial Science and Technology Department Seedling Project (MZGC20240071 & MZGC20240072)
  • Sichuan Provincial Healthcare Commission Popularization and Application Project (21PJ194)
  • Scientific Research Project of Nanchong Science and Technology Bureau (23JCYJPT0028)
  • the Natural Science Foundation of China (82203851)
  • Doctoral Fund of North Sichuan Medical College (CBY22-QDA01)

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